Protein Protocols Protein Protocols

Peptide Conjugation and Immunization 957
conjugation procedures. This intermediate then reacts with the primary amine to form
an amide derivative. Failure to immediately react with an amine results with hydrolysis
of the intermediate. Furthermore, hydrolysis of CDI itself is a competing reaction during
the coupling and is dependent on temperature 4-morphylinoethansulfonic acid
(MES) can be used as an effective carbodiimide reaction buffer in place of water. Phosphate
buffers reduce the efficiency of the CDI reaction, although this can be overcome
by increasing the amount of CDI used to compensate for the reduction and efficiency.
Loss of efficiency of the CDI reaction is even greater with Tris, glycine, and acetate
buffers, and therefore use of these these should be avoided.
2.3.2. Glutaraldehyde
Glutaraldehyde links primary amino groups (either the N-terminal or lysyl residues)
on both the peptide hapten and the carrier. This linkage allows free rotation of the
hapten, which reduces possible steric hindrance which may otherwise block access to
the immune system by the large carrier molecule.
2.3.3. Sulfo-succinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxylate (sulfo-SMCC)
A peptide with a free sulfhydryl group, such as a synthetic peptide with a terminal
cysteine residue, provides a highly specific conjugation site for reacting with sulfo-
SMCC. This cross-linker contains a maleimide group that reacts with free sulfhydryl
groups, along with an N-hydroxysuccinimidyl ester group that reacts with primary
amines (2). All peptide molecules coupled using this chemistry display the same basic
antigenic conformation. They have a known and predictable orientation leaving the
molecule free to interact with the immune system. This method can preserve the major
epitopes on a peptide while enhancing the immune response. The water solubility of
sulfo-SMCC, along with its enhanced maleimide stability, makes it a favorite for hapten
carrier conjugation.
2.3.4. Bis-Diazotized Benzidine
Bis-diazonium salts bridge tyrosyl or hystidyl residues between the hapten and carrier.
Overnight treatment of benzidine at 4°C with nitrous acid (hydrochloric acid and
sodium nitrite) results in the two amino groups being diazotized. These two diazonium
groups allow coupling at both ends of the molecule. Although limited in coupling
points, diazotized benzidine provides a spacer arm holding the hapten away from the
carrier and usually results in an excellent antigenic response.
2.4. Adjuvants
The adjuvant is important for inducing an inflammatory response. The author has
had continuous success over two decades using Freund’s adjuvant. Various synthetic
adjuvants are available such as AdjuPrime (Pierce) and Ribi Adjuvant System (Ribi
Immunochemicals) (7). The author has had very limited success with the latter adjuvant,
when used to immunize with several different small peptides. In contrast,
responses with Freund’s adjuvant run in parallel have always been good.
The conjugate is injected in the form of an emulsion made with Freund’s adjuvant,
which is a mixture of one part of detergent (Arlacel A, Sigma Chemicals) with four
parts of n-hexadecane. This permits slow release of the coupled hapten into the circu-